1. Field of the Invention
The present invention relates to the field of nucleic acid synthesis and analysis, and particularly to the field of preparing single stranded DNA probes or primers of defined sequence and length.
2. Related Art
The generation of single-stranded DNA has a large number of applications in understanding biological functions of gene expression and function, treatment of diseases in plants and animals, and in applications to diagnostics and forensics. There are currently several applications that rely on the use of long oligonucleotides as probes. These include molecular inversion probes (Willis et al., 2000 U.S. Pat. No. 6,858,412), wherein probes termed “pre-circle” probes are hybridized at either end to a target, then circularized by filling the gap between the ends. It is said that the gap may be between 1 and 2000 nucleotides (Col. 14 1.35), but the examples are directed to single nucleotide gaps. This method is based on the fact that the two targeting domains of a pre-circle probe can be preferentially ligated together, if they are hybridized to a target strand such that they abut and if perfect complementarity exists at the two bases being ligated together. Perfect complementarity at the termini allows the formation of a ligation substrate such that the two termini can be ligated together to form a closed circular probe. If this complementarity does not exist, no ligation substrate is formed and the probes are not ligated together to an appreciable degree. Once the precircle probes have been ligated, the unligated precircle probes and/or target sequences are optionally removed or inactivated. The closed circular probe is then linearized by cleavage at the cleavage site, resulting in a cleaved probe comprising the universal priming sites at the new termini of the cleaved probe. The patent further states that, due to the length of the precircle probes, it is preferred that each target domain range in size from about 5 bases to about 100 bases, with from about 5 to about 40 being especially preferred.
Padlock probes are described in Landegren et al., U.S. Pat. No. 6,235,472, and Landegren et al., 2001). The term “padlock probe” refers to a probe designed to be circularized in the presence of a target sequence, so that it may be caused to close around the target-containing nucleic acid strand such that the cyclic probe will interlock with and thereby be efficiently linked to the target nucleic acid to be detected. In other words, because of the helical nature of double-stranded nucleic acids, such as DNA, circularized probes will be wound around the target strand, topologically connecting probes to target molecules through catenation, in a manner similar to “padlocks”. Such covalent catenation of probe molecules to target sequences results in the formation of a hybrid that resists extreme washing conditions, serving to reduce non-specific signals in genetic assays. Any probes hybridizing in a non-specific manner may therefore be efficiently removed by subjecting the target to non-hybridizing conditions and/or exonuclease activity. Further, the novel method may be performed with even very short synthetic probes since only part of the probe molecule needs to form a rigid double-stranded DNA molecule with the target molecule, whereas the rest of the probe molecule may be highly flexible, optionally branched single-stranded DNA or any other spacer material. In this system, a probe is hybridized to a target nucleic acid sequence, such as a DNA strand, via two end segments of the detecting reagent, designated Probe 1 and Probe 3, the latter being complementary to two respective non-contiguous sequences of the target molecule. An additional probe, designated Probe 2, is hybridized to the intermediate segment of the target molecule with the probe ends in juxtaposition to Probe 1 and Probe 3, respectively, and then ligated to the two ends.
Another application of single stranded DNA molecules is described in Fredriksson S., et al., “Protein detection using proximity-dependent DNA ligation assays,” Nat Biotechnol, 2002 May; 20 (5):473-7. This paper describes a technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein.
Another method for nucleic acid formation is strand displacement amplification (SDA), which is generally described in U.S. Pat. Nos. 5,455,166 and 5,130,238. A single stranded target nucleic acid, usually a DNA target sequence, is contacted with an SDA primer. An “SDA primer” generally has a length of 25-100 nucleotides and is substantially complementary to a region at the 3′ end of the target sequence, and the primer has a sequence at its 5′ end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to herein as a “nicking enzyme” or a “nicking endonuclease”, which is chosen to cleave a strand either at the recognition site, or either 3′ or 5′ to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides.
For many of the assays described above, single stranded DNA probes are synthesized chemically. Currently, these probes are very expensive to manufacture to the required specificity and purity that these applications demand.
Various attempts have been made to produce defined single-stranded DNA. Nikiforov and Knapp (U.S. Pat. No. 5,518,900) describe a method for producing single-stranded DNA from a PCR fragment where one of the primers used for amplification has a modification that makes that strand resistant to exonuclease digestion. This method suffers from the drawback that every probe requires the synthesis of oligonucleotides with chemically modified nucleotides, which is not economically feasible in large-scale genomic studies.
Higuchi et al., (1989) describe a method for producing single-stranded DNA from PCR fragments where one of the amplification primers is phosphorylated and the corresponding strand with the phosphorylated primers is a preferential substrate for nuclease digestion. The drawback of this method is that the non-phosphorylated strand from a blunt-end DNA molecule (as in a PCR product) acts as a substrate, though with reduced efficiency.
Binkowski, et al., “Correcting errors in synthetic DNA through consensus shuffling,” Nucleic Acids Res, Mar. 30, 2005; 33 (6): e55, describe a method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS).